The present invention relates generally to polyaromatic compounds and methods for their production and more particularly to the synthesis of polyaromatic compounds using biocatalysts.
Traditionally, phenolic resins, such as novolacs and resoles, have been commercially prepared by condensing phenol and formaldehyde at various desired molar ratios in the presence of a particular acid or base catalyst. However, due to the carcinogenic nature of formaldehyde, the use of formaldehyde in phenolic resin production poses a major threat to the health and safety of personnel involved therein. In addition to the aforementioned health and safety risks associated with the use of formaldehyde in phenolic resin production, residual amounts of formaldehyde are typically present in the finished product--a result that is both undesirable and largely unavoidable.
One alternative to using formaldehyde to prepare phenolic resins has been to use inorganic catalysts (e.g., copper halide+an aliphatic amine) or biocatalysts (e.g., peroxidase enzymes). In addition to avoiding the problems associated with the use of formaldehyde, phenolic polymers produced by enzymatic reactions typically have the additional advantage of having extensive backbone conjugation, thereby leading to conductivity under doped conditions. Other advantages of using enzymes to catalyze phenol polymerization include mild reaction conditions, fast reaction rates, high substrate specificity and minimal by-product formation. Horseradish peroxidase is the most commonly used enzyme for these polymerization reactions, which are typically carried out in solvent/water mixtures and microemulsions. Polymer molecular weight and polydispersity are controllable using the aforementioned enzymatic method.
One shortcoming that has been noted in connection with the aforementioned phenolic resins is that they have limited solubility in water. As a result, such resins are limited in their utility in certain applications, such as in use in rechargeable batteries, photolithography, fire retardants and detergents. For example, polypyrrole is the most widely used conjugated polymer in enzyme-mediated glucose detection. Polythiophene, polydiacetyline and polyaniline are a few other polymers used in this context. However, these polymers lack the flexibility to incorporate recognition sites into their backbone and/or an ease of synthesis and/or modification. Polypyrroles, for example, are usually obtained as water-insoluble films which are used to physically trap or to sandwich biomolecules in the preparation of biosensing elements. Incorporation by covalently linking the biomolecules to the polymer backbone requires the use of functionalized monomers with carboxyl or amine groups. Such functionalized thiophene monomers have been used to develop a generic molecular assembly for pesticide sensor applications.
In U.S. Pat. No. 5,508,180, inventors Johnson et al., which issued Apr. 16, 1996, and which is incorporated herein by reference, there is disclosed a biocatalytic oxidative process for preparing phenolic resins using soybean peroxidase. According to said patent, phenols which are preferred for polymerization are represented by the following formula: ##STR2## wherein Y and Z are selected from the group consisting of a hydrogen atom, a halogen atom, an alkyl group, an alkoxy group, an aryl group, an allyl group, a phenylalkyl group, a --COOR group, a --NR.sup.1 R.sup.2, where R represents a hydrogen atom or a lower alkyl group, and R.sup.1 and R.sup.2 represent a hydrogen atom, an alkyl group, or a phenylalkyl group or Z in conjunction with the adjacent meta position forms a condensed benzene ring. Since polymerization proceeds via the ortho or para positions, when Y is at the ortho or para position, at least one of Y and Z must be a hydrogen atom or Z must form said condensed benzene ring. Y is preferably para to the phenolic hydroxyl group. Otherwise, the phenol adds as a terminal group as discussed below. At the para position, long chain alkyl groups have a tendency to slow the reaction. The reaction appears to proceed best when Y is p-phenyl, p-methoxy or p-halogen. A single phenol or a mixture of phenols may be used in the polymerization reaction. In certain applications, it may be desirable to produce phenolic resins having certain terminal groups.
In U.S. Pat. No. 5,420,237, inventors Zemel et al., which issued May 30, 1995, and which is incorporated herein by reference, there is disclosed a method for enzymatically synthesizing electrically conductive and substituted and unsubstituted polyanilines. According to said patent, aniline monomer(s), an oxidizing agent, which comprises an enzyme and an electron acceptor, and an acidifying agent are reacted together to form polyanilines. The patent identifies the following as illustrative of such aniline monomers: ##STR3## wherein n is an integer from 0 to 4; m is an integer from 1 to 5 with the proviso that the sum of n and m is equal to 5 and that at least one position on the aniline ring is a moiety which allows oxidative coupling at that position; R.sub.1 is a hydrogen or a permissible R.sub.2 substituent; and R.sub.2 is the same or different at each occurrence and is selected from the group consisting of alkyl, alkenyl, alkoxy, cycloaklyl, cycloalkenyl, alkanoyl, alkylthio, aryloxy, alkylthioalkyl, alkylaryl, arylalkyl, amino, alkylamino, dialkylamino, aryl, alkylsulfinyl, aryloxyalkyl, alkylsulfinylalkyl, alkoxyalkyl, alkylsulfonyl, aryl, arylthio, alkylsulfonyl, carboxylic acid, hydroxy, halogen, cyano, sulfonic acid, nitro, mercapto, alkylsilane or alkyl substituted with one or more sulfonic acid, carboxylic acid, halo, nitro, mercapto, cyano, or epoxy moieties; or any two R.sub.2 groups together may form an alkylene or alkenylene chain completing a 3, 4, 5, 6 or 7 membered aromatic or alicyclic ring, which ring may optionally include one or more divalent nitrogen, sulfur, sulfonyl, ester, carbonyl, sulfonyl, or oxygen atoms, or R.sub.2 is an aliphatic moiety having repeat units of the formula: EQU --(OCH.sub.2 CH.sub.2)qO-- or --(OCH.sub.2 CH(CH.sub.3))qO--
in which q is a positive whole number. A moiety which allows oxidative coupling is any moiety that does not hinder the head-to-tail coupling of the monomers in forming polyaniline. An example of such a moiety is hydrogen or deuterium.
In Ayyagari and Akkara, "Enzymatic synthesis of multifunctional polyphenols for biosensor applications," Polymeric Materials Science & Engineering, 76:610-611 (1997), which is incorporated herein by reference, there is disclosed the copolymerization of p-ethylphenol (EP) and 3-(4-hydroxyphenyl)propionic acid (HPPA) at different molar ratios. According to the publication, the copolymerization reaction involves dissolving the monomers in ethanol and adding HEPES buffer (1 mM, pH 7.5) to obtain a final ethanol content of about 25% (v/v). The final monomer concentration is 0.15M. An aliquot of enzyme solution, prepared by dissolving horseradish peroxidase (Type II) in N-[2-hydroxyethyl]piperazine-N'[2-ethanesulfonic acid] (HEPES) buffer at a known concentration, is added to the monomer solution such that the final enzyme concentration is 0.1 mg/ml. The reaction is initiated by dropwise addition of H.sub.2 O.sub.2. The total amount of H.sub.2 O.sub.2 added is 30% in excess of stoichiometric amount. The reactions are continued for 24 hours. At the end, solvent is evaporated under the flow of nitrogen at room temperature. The dry material is washed with isooctane and water separately to remove unreacted monomers, salts and the enzyme. The material is then dried at 40.degree. C. under vacuum.
Additional patents of interest include the following, all of which are incorporated herein by reference: U.S. Pat. No. 5,639,806, inventors Johnson et al., issued Jun. 17, 1997; U.S. Pat. No. 5,606,010, inventors Erhan et al., issued Feb. 25, 1997; U.S. Pat. No. 5,367,043, inventors Butler et al., issued Nov. 22, 1994; U.S. Pat. No. 5,278,055, inventors Cyrus, Jr. et al., issued Jan. 11, 1994; U.S. Pat. No. 4,900,671, inventors Pokora et al., issued Feb. 13, 1990; U.S. Pat. No. 4,882,413, inventor Erhan, issued Nov. 21, 1989; and U.S. Pat. No. Re. 35,247, inventors Cyrus, Jr. et al., reissued May 21, 1996.